While saccharride is a major component constituting a living body together with nucleic acid and protein, its structure and function are not known as clearly as those of nucleic acid and protein. Carbohydrate generally forms a polymer having sugar chains in sequence and binds with protein and lipid to form a pronouncedly complicated complex molecule generally referred to as a glycoprotein, glycolipid or proteoglycan. While nucleic acid and protein are polymers wherein constituent units of nucleotide and amino acid are linearly bonded, the structure of the sugar chain is far more complicated than nucleic acid and protein, as its molecule contains plural branches and the constituent unit monosaccharide binds in a variety of manners. Such complicated structure of the saccharide is one of the major causes of the delay in the study of saccharide.
Along with the elucidation of the saccharide's role in cell recognition, immunity, differentiation, fertilization, senescence, malignant alteration and the like in recent years, saccharide has become a target of study that attracts significant attention. Under the circumstances, many attempts have been made to synthesize a sugar chain having a natural structure or a novel sugar chain. As regards nucleic acid and protein, automatic synthesis techniques have been already established, and these techniques have obviously accelerated the progress of the researches in these fields. An automatic synthesis technique for a sugar chain has been eagerly desired, but effective methods of various protection and deprotection and a method to achieve a high yield, steroselective glycosylation reaction have not been developed to satisfaction, and thus, the desired technique has not been available.
Recently, Danishefsky et al. proposed a solid phase synthetic method of saccharide utilizing glycal (Science, 260, 1307 (1993)), and this has led to the resolution of the problems as to the high yield and steroselective glycosylation to a certain extent. This method comprises (i) binding a glycal to a polystyrene-divinylbenzene copolymer via a diphenylsilyl group to allow reaction between said glycal and 3,3-dimethyldioxirane, that converts glycal to a 1,2-anhydrosugar, and (ii) using this anhydrosugar as a sugar donor, reaction with a different glycal suitably protected to form a glycoside glycal, and these steps are repeated. According to this method, a new glycosidic linkage can be stereoselectively formed, but only a compound that has a glycosidic linkage at the trans-position with respect to the 2-position hydroxy of the donor can be formed.
Meanwhile, a solid phase synthetic method of a sugar chain has been proposed, that utilizes glycosyltransferase capable of stereoselectively forming a glycosidic linkage without any protection. This method has not been further developed, due to the fact that the available glycosyltransferase is limited in kind and is expensive. In recent years, however, genes of various glycosyltransferases have been isolated and a large-scale production of glycosyltransferase by genetic recombination techniques has become possible.
For example, U. Zehavi et al. have reported on a solid phase synthesis method by glycosyltransferase using a polyacrylamide gel bound with an aminohexyl group as a solid phase carrier (Carbohydr. Res., 124, 23 (1983), Carbohydr. Res., 228, 255 (1992)). This method comprises the steps of converting a suitable monosaccharide to 4-carboxy-2-nitrobenzylglycoside, condensing this glycoside with amino group of the above-mentioned carrier, elongating the sugar chain by glycosyltransferase using the condensate as a primer, and releasing the oligosaccharide by photolysis. According to this method, however, sugar transfer yield is low and is less than 10%.
It has been a common understanding that glycosyltransferase does not react well with saccharide or oligosaccharide bound to a solid phase carrier, and efficient elongation of sugar chain is difficult to achieve. A recent report has documented that linkage between 4-carboxy-2-nitrobenzylglycoside and solid phase carrier by a linker having a long chain, such as hexamethylene and octamethylene, led to an improved sugar transfer yield at the maximum of 51% (React. Polym., 22, 171 (1994), Carbohydr. Res., 265, 161 (1994)).
C. -H. Wong et al. have documented a report wherein a sugar chain is elongated using a glycosyltransferase and aminated silica bound with the following group of (a) as primers, and the elongated sugar chain is cleaved out utilizing hydrolysis of .alpha.-chymotrypsin (J. Am. Chem. Soc., 116, 1136 (1994)). By this method, the transglycosylation yield was 55%. C. -H. Wong et al. revised the group to be bonded to the solid phase carrier to the following (b) and reported a method wherein the sugar chain was elongated by glycosyltransferase and released by hydrazinolysis, whereby the transglycosidation proceeded almost quantitatively (J. Am. Chem. Soc., 116, 11315 (1994)). ##STR1## wherein Boc is t-butoxycarbonyl and Ac is acetyl. ##STR2## wherein Ac is acetyl.
As another method, M. Meldal et al. have reported a method comprising elongation of sugar chain using glycosyltransferase and, as a primer, a polymer of mono- and diacryloyl compound of diaminated poly(ethylene glycol), the polymer having a group of the following formula (c) bonded thereto, and release of the sugar chain by trifluoroacetic acid, wherein the transglycosidation proceeded almost quantitatively (J. Chem. Soc., Chem. Commun., 1849 (1994)). ##STR3## wherein Ac is acetyl.
As mentioned above, when sugar chain is elongated by glycosyltransferase on a solid phase carrier, the kind of group (linker) which connects the solid phase carrier to the sugar residue (receptor of initial transglycosylation) varies transglycosylation yield. When the sugar chain is liberated from the carrier, the presence of a specifically cleavable bond in the linker is markedly advantageous.
In sugar chain elongation by glycosyltransferase, the use of an immobilized glycosyltransferase that permits repetitive use is desirable, since glycosyltransferase is highly expensive, though a large-scale production by genetic recombination techniques is becoming available. It should be noted that the above-mentioned methods are associated with a drawback that an immobilized glycosyltransferase cannot be used due to the sugar chain elongation on an insoluble carrier.
It is necessary to carry out the sugar chain elongation not on an insoluble carrier but on a water soluble carrier, if an immobilized glycosyltransferase is to be used.
As a sugar chain synthetic method using a water soluble carrier, the present inventors have reported a method comprising elongation of a sugar chain using a glycosyltransferase and, as a primer, a polyacrylamide having a group of the following formula (d) bonded to the nitrogen atom of the amide moiety, and cleavage of the elongated sugar chain by hydrolysis of .alpha.-chymotrypsin (Tetrahedron Lett., 35, 5657 (1994)). However, the glycosyltransferase also used in this method is a soluble enzyme and is not immobilized. Thus, there has been found no precedent case where a sugar chain was synthesized by an immobilized glycosyltransferase and a water soluble carrier. ##STR4## wherein Ac is acetyl.
According to C. Auge, for immobilization of a glycosyltransferase, galactosyltransferase is first immobilized on an agarose gel activated by cyanogen bromide, and oligosaccharide is synthesized (Pure & Appl. Chem., 59, 1501 (1987)). According to J. Thiem et al., galactosyltransferase is immobilized on aminopropyl silica using glutaraldehyde, and used for the synthesis of oligosaccharide (Angew. Chem. Int. Ed. Engl., 25, 1096 (1986)). However, the substrate is a typical monosaccharide or oligosaccharide in both cases, and transglycosylation to a polymer substrate having sugar chains in sequence with immobilized glycosyltransferase has not been reported. Generally, an immobilized enzyme is considered to be inferior to a soluble enzyme in the reactivity with a polymer substrate. This is attributable to the difficulty experienced by an immobilized enzyme in contacting a polymer substrate, in comparison to a soluble enzyme. Hence, an efficient elongation reaction of a sugar chain on a water soluble polymer substrate (carrier) using an immobilized glycosyltransferase will be extremely useful.
It is therefore an object of the present invention to provide a method for synthesizing a sugar chain using an immobilized glycosyltransferase.